Project A: Establish the role of factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, in root formation and cementogenesis and apply this knowledge to deliver factors locally/systemically to regenerate periodontal tissues, using rodent models of periodontal disease. Results demonstrate the importance of Pi/PPi and SIBLING family genes/proteins during mineralization, highlighting the need for both physiochemical and cellular molecular factors to achieve homeostatic balance required for formation/regeneration of periodontal tissues. 1. Cell, Tissues and Animal Models of Periodontal Disease: a. ANK, NPP1 and TNAP in vivo and vitro: Examining Ank and Npp1 KO animals demonstrated a compensatory mechanism related to gene expression. Also, the expression patterns and functions of TNAP and NPP1 during cementogenesis suggest that early expression of TNAP creates a low PPi environment allowing for AEFC initiation, while later expression of NPP1 increases PPi, restricting AEFC apposition. These results provide the rationale for in vivo studies, started summer 2017, using periodontal wound healing models in rodents to deliver factors controlling Pi/PPi, using lentivirus constructs and scaffolds. b. Ank, Enpp1 double KO mice and cells, in vitro: To determine the mechanisms by which ANK and ENPP1 regulate cementogenesis - e.g., are their effects additive? synergistic? and are there mechanistic differences in how they control cementogenesis? To address these questions, we established Ank, Enpp1 dKO mice. Results to date indicate that acellular cementum phenotype exhibited by the Ank, Enpp1 dKO is a composite phenotype of the single KO, suggesting that ANK and ENPP1 have non-redundant roles in upregulating extracellular PPi. Further, we are contrasting periodontal wound healing capabilities of Ank vs Enpp1 KO mice. Ongoing studies, using proteomics, PCR microarray and RNA seq technologies, are defining protein and gene expression profiles of PDL tissues obtained from single and double KO mice at various stages of tooth root development. Ank KO, Enpp1 KO and Ank, Enpp1 dKO cells are being generated using CRISPR-CAS technologies. c. Orthodontic tooth movement, using an Enpp1 KO mouse model: Predictable regeneration of periodontal tissues is a major unrealized goal of periodontal therapy, including for orthodontic-associated tooth root resorption. Therefore there is a need to better understand the factors regulating formation and regeneration of cementum. Studies have revealed that cementum is exceptionally sensitive to regulation by inorganic pyrophosphate (PPi). Local PPi concentrations are determined by PPi regulators including ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), which increases PPi concentrations by ATP hydrolysis. Using 60 days postnatal Enpp1 knockout (Enpp1-/-) mice, featuring reduced PPi and increased cervical cementum, we employed a model of orthodontic tooth movement using a stretched closed coil spring ligated between maxillary left first molar and maxillary incisors. Tooth movement, osteoclast response, and tooth root resorption were evaluated by micro-computed tomography, histology, and histomorphometry. Altered response to orthodontic loading, decreased tooth movement, and altered osteoclast/odontoclast distribution in the Enpp1-/- mouse molars provide novel insights into the intersection of mineral metabolism and periodontal remodeling. Wolf M, Ao M, Chavez MB, Thumbigere-Math V, Becker M, Chu EY, Jger A, Somerman MJ, Foster BL. Reduced Orthodontic Tooth Movement in Enpp1 Mutant Mice with Hypercementosis. JDR, 2017. In review. Project B: Defining the role of extracellular matrix proteins in periodontogenesis with a focus on SIBLINGS and collagen. 1. BSP and OPN: BSP and OPN belong to the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family, a family of acidic phosphoproteins expressed in mineralized tissues. We reported that BSP KO mice have a profound periodontal phenotype, similar to Alpl KO mice. We identified OPN as a factor of interest based on proteomic analysis of PDL from Ank-/- mice. Several models were used to determine OPN's function within the PDL region. We conclude that OPN does not have a non-redundant or critical role in regulating cementum mineralization and periodontal structure and function during development and yet, in wound healing models, loss of OPN function may have consequences, which is being explored by several groups at this time. Foster BL, Ao M, Salmon CR, Chavez MB, Kolli TN, Tran AB, Chu EY, Kantovitz KR, Yadav M, Narisawa S, Milln JL, Nociti Jr. FH, Somerman MJ. Osteopontin Regulates Dentoalveolar Mineralization. Bone, 2017. In Review. To further define the mechanistic aspects of BSP's regulation of genes associated with Pi/PPi modulation, Bsp KO cementoblasts (OCCM30) were generated using CRISPR/CAS technology. Preliminary results suggest that BSP may have at least two roles in the mineralization process, i.e., regulating modulators of local Pi/PPi levels and regulating expression of transcription factors through various feedback loops (Ao et al., 2017). In addition, we are developing an Opn x Bsp dKO to determine if loss of OPN expression would alter the periodontal phenotype reported for the Bsp KO mice. 2. Collagen: Logic would suggest that individuals with osteogenesis imperfecta (OI), a heritable disorder of the extracellular matrix characterized by low bone mass, brittle and fragile bones, and bone fractures, would have marked periodontal defects. Yet, reports to date focus on general bone pathology and in some cases, dentin defects (dentinogenesis imperfecta (DI)), with limited attention to the periodontium. A multi-collaborative project with several extramural investigators as part of the Brittle Bone Consortium has enabled us to obtain tissues from four types of OI mice as well as extracted teeth from OI patients. For the first time, we identify periodontal changes in mice resulting from autosomal recessive and autosomal dominant OI. Changes reflect developmental defects, as well as altered function and remodeling during later stages in life. Chu et al., plan to submit Sept. 2017. Other projects: IRF6, CL/P and dental manifestations: Emily Chu DDS, PhD for fulfillment of her PhD. (Not continued at NIAMS/NIH). Chu EY et al. Full spectrum of Postnatal Tooth Phenotypes in a Novel Irf6 Cleft Lip Model. J Dent Res 95(11):1265-73, 2016.